Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs.

With high throughput screening, novel therapeutic agents can be efficiently identified. Unfortunately, researchers only resort to in vitro cell viability assays for screening of anticancer drugs for retinoblastoma, the most common intraocular cancer in the childhood. Current available animal models of retinoblastoma require more than 2 weeks for tumour formation and the investigation of the efficacy of therapeutic agents. In this study, we established a novel orthotopic transplantation model of retinoblastoma in zebrafish as an in vivo animal model for screening of anticancer drugs. We injected retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours after fertilization. Eyeballs of zebrafish were scanned daily under the confocal laser microscope, and the tumor population was quantitatively analyzed by measuring the mean intensity of green fluorescent protein (GFP). Transplanted retinoblastoma cells were isolated to perform further analyses including Western blotting and reverse transcriptase-polymerase chain reaction to confirm that retinoblastoma cells maintained their characteristics as tumor cells even after transplantation and further isolation. To figure out the potential of this model for screening of anticancer drugs, zebrafish were cultured in Ringer's solution containing carboplatin and melphalan after the injection of retinoblastoma cells. The degree of the tumor population was dependent on the number of retinoblastoma cells injected and maintained stably for at least 4 days. Transplanted retinoblastoma cells maintain their proliferative potential and characteristics as retinoblastoma cells after isolation. Interestingly, systemic application of carboplatin and melphalan demonstrated significant reduction in the tumor population, which could be quantitatively analyzed by the estimation of the mean intensity of GFP. This orthotopic retinoblastoma model in zebrafish is expected to be utilized for the screening of anticancer drugs for the treatment of retinoblastoma.